The demand for less hazardous and environmentally friendly surface disinfectants is growing and essential oils may be efficient alternatives. Vapor phase assay is a promising technique which explore the volatility of essential oils for determining the antimicrobial activity. In this study, we modified the conditions of the inverted Petri dish technique to evaluate the minimum inhibitory concentrations (MIC). As a proof of concept, the antimicrobial activity of Hesperozygis myrtoides essential oil (HM) was studied for the first time in the vapor phase. According to the established protocol the oil volatilization dynamics was used to determine the major compounds in the vapor phase. After 270 min, the relative percentage was like that observed in the crude oil analysis. The HM was also evaluated for safety (IC50=921.2 µg ml-1), being considered safe in case of acute oral exposure. The oil vapors completely inhibited the growth of Staphylococcus aureus (MIC=0.39 mg l-1), Candida albicans (MIC=0.83 mg l-1), Aspergillus brasiliensis (MIC=378 mg l-1) and Bacillus subtilis (MIC=704.8 mg l-1). This protocol offers an economical and efficient alternative for antimicrobial activity and safety determination of essential oils, can be used for the evaluation of several applications of essential oils, as disinfectant agent in inhabited environments.